Lactic acid and its salts have long been utilized in a wide variety of applications in the chemical, cosmetic, food and pharmaceutical industries. More recently, new bioengineering materials based on lactate, such as biodegradable lactide polymers, have kindled an increased demand for lactate and especially for the free acid form of either L- or D-lactate. The use of lactic acid in the production of various industrial polymers has been described, for example, in U.S. Pat. Nos. 5,142,023; 5,247,058; 5,258,488; 5,357,035; 5,338,822; 5,446,123; 5,539,081; 5,525,706; 5,475,080; 5,359,026; 5,484,881; 5,585,191; 5,536,807; 5,247,059; 5,274,073; 5,510,526; and 5,594,095. (The
complete disclosures of these seventeen patents, which are owned by the assignee of the present application, Cargill, Inc. of Minneapolis, Minnesota, are incorporated herein by reference.)
While chemical processes can be used to produce lactic acid, the rising cost of petrochemical feedstocks and the need to resolve the racemic lactate mixture produced by conventional chemical methods, make fermentation methods an attractive alternative for the manufacture of lactate enriched in one of its optical isomers. The processes used to produce biodegradable lactide polymers typically require the free acid form of either L- or D-lactate as a starting material. Unfortunately, as with most organic acid fermentations, the end-product inhibition by the organic acid (lactic acid in this instance) can be a major obstacle to efficient fermentation. Bacterial strains typically employed in lactate fermentations may be inhibited by low pH in addition to lactate concentration. To overcome this problem, industrial lactate fermentation processes are typically run at a higher pH, e.g., at least about 5.0 and often at or above 6.0. This results in the production of a lactate product which is essentially all present in the form of a salt. Additional process step(s) are typically required to remove the cationic counterion and isolate the desired free lactic acid. Moreover, since high concentrations of certain salts, e.g., sodium cations, may have an inhibitory effect on fermentation, the type and/or amount of salt present can also influence the efficiency of the fermentation.
The production of racemic lactate from enzyme-thinned corn starch using lactobacillus amylovorus has been reported. While relatively high production levels at pH as low as 4.2 have been reported, this fermentation does not provide lactate enriched in either optical isomer.
A number of approaches for improving the efficiency of lactate fermentations have been reported. Several of these involve removal of free lactic acid from the fermentation broth on a continuous basis. For example, electrodialysis has been used to reduce the end product inhibition through removal of lactate from the fermentation broth. The high cost of dialysis membranes coupled with a low lactate gradient has generally lowered the attractiveness of this approach. Ion exchange and the use of polyvinylpyridine to remove lactate from the fermentation medium have also been reported. Yet another method which was described recently, involves a multistage extraction procedure. This process involves an extraction of lactate from the broth with a tertiary amine in an attempt to keep the broth pH from dropping to a value which inhibits further lactate production. The lactate production levels reportedly achieved via this method are still, however, quite low. Utilization of this method may also require that the extracted fermentation broth be subjected to a second extraction to at least reduce the residual concentration of tertiary amine extractant before recycling the extracted broth back into the fermentation reaction.
All of these approaches to producing lactic acid in its free acid form based on fermentation of lactobacillus suffer from one or more disadvantages. Alternative approaches based on the fermentations of other more acid tolerant microorganisms have also been reported. Yeasts, such as Saccharomyces cerevisiae, are capable of growth at much lower pH than lactobacillus. Recombinant yeast strains have been produced by introducing the lactate dehydrogenase gene from a bacterial (lactobactobacillus) or mammalian (bovine) source into Saccharomyces cerevisiae. The recombinant yeast strains are reportedly able to produce lactate at or below the pKa of lactic acid (about 3.8). Ethanol is, however, the major fermentation product generated by the these recombinant yeast strains. This both lowers the efficiency of lactate production and introduces additional potential issues with regard to the separation and purification of free lactic acid. Lactic acid production by a pellet form of the fungus, Rhizopus orgyzae, has also been reported. This fungal fermentation also typically produces glycerol and/or ethanol as major byproducts. The yield of free lactic acid was optimized in this instance by continuous removal from the fermentation broth using a polyvinylpyridine (xe2x80x9cIPVPxe2x80x9d) column. No lactate concentrations higher than about 25 g/L were reported to have been generated using the Rhizopus/PVP method.
The present invention relates to the production of lactate via fermentation. It particularly concerns fermentation with acid-tolerant bacteria to produce a fermentation broth with high levels of free lactic acid. The presence of the high level of free lactic acid can facilitate the down stream processing required to isolate lactate in its free acid form from the broth.
The process provided herein for producing lactic acid includes incubating acid-tolerant bacteria, such as acid-tolerant homolactic lactobacillus, in nutrient medium at a pH which furnishes a substantial portion of the lactate product in the free acid form. Herein, when the term xe2x80x9cacid-tolerantxe2x80x9d is employed in reference to bacteria, the intent is to refer to bacteria which are capable of producing lactate at a pH sufficient to furnish a substantial portion of the lactate product in the free acid form. The acid-tolerant bacteria are typically capable of producing at least about 25 g/L free lactic acid. Such bacteria generally can also produce at least about 50 g/L lactate in nutrient medium at an xe2x80x9caverage incubation pHxe2x80x9d of no more than about 4.2.
If fermentation is not carried out to a point where the limiting lactate concentration is reached, the xe2x80x9caverage incubation pHxe2x80x9d is determined based on an average of the pH values measured at ten(10) or more equal time intervals over the course of the fermentation. The present fermentation process may be run in a continuous fashion. Under such conditions, steady state conditions (in terms of pH, lactate concentration and nutrient concentrations) are generally achieved and maintained after an initial startup phase has been concluded. When fermentation is conducted in this manner, the average incubation pH is the average pH of the broth after the initial startup phase has been completed, i.e., the pH during the startup phase is ignored in determining the average incubation pH.
If fermentation is carried out to a point where pH and/or lactic acid concentration inhibits further lactate production, the xe2x80x9caverage incubation pHxe2x80x9d is determined based on an average of the pH values measured at ten(10) or more equal time intervals over the time period necessary to produce 90% of the limiting lactate concentration. As used herein, the xe2x80x9climiting lactate concentrationxe2x80x9d is the lactate concentration under a given set of incubation conditions (nutrient medium, temperature, degree of aeration) at which pH and/or lactic acid concentration generated by the fermentation inhibits further lactate production. As used herein, the term xe2x80x9climiting incubation pHxe2x80x9d means the pH of the fermentation broth for a given set of incubation conditions at which the pH and/or lactic acid concentration inhibits further lactate production. Inhibition of lactate production is considered to have occurred when the amount of lactate produced does not increase by more than about 3% upon further incubation for a period of up to about twelve (12) hours under the same conditions. This definition presumes that sufficient nutrients for lactate production are still available in the fermentation broth.
Herein the terms xe2x80x9cnutrient mediumxe2x80x9d and xe2x80x9cfermentation brothxe2x80x9d are used interchangeably. These terms refer to both (i) media in the form originally provided to the acid-tolerant bacteria as a source of nutrient and (ii) media produced after some or all of the originally provided nutrients have been consumed and fermentation products including lactate have been excreted into the media by the bacteria.
In the present process, the pH of the fermentation broth after incubation of the acid-tolerant bacteria to produce lactate is typically no more than about 4.2 (xe2x80x9cfinal incubation pHxe2x80x9d). As referred to herein, the xe2x80x9cfinal incubation pHxe2x80x9d is the pH of the fermentation broth at the point that growth and/or lactate production by the acid-tolerant bacteria ceases. The cessation of growth and/or lactate production may be the result of a change in reaction temperature, the exhaustion of one or more necessary nutrients in the fermentation broth, a deliberate change in pH, or the separation of the fermentation broth from the bacterial cells. In those instances where fermentation is arrested by the addition of sufficient acid or base to the broth to stop lactate production, the final incubation pH is defined to be the pH of the nutrient medium just prior to the addition. Alternatively, growth and/or lactate production may stop due to the accumulation of one or more fermentation products and/or a change in broth pH resulting from the accumulation of fermentation products, i.e., the fermentation reaction has reached a self limiting point for the given set of incubation conditions. As noted above, it is quite common for bacterial fermentations which produce an organic acid such as lactic acid to be subject to end-product inhibition.
The term xe2x80x9clactatexe2x80x9d as used in this application refers to 2-hydroxypropionate in either its free acid or salt form. The terms xe2x80x9clactic acidxe2x80x9d and xe2x80x9cfree lactic acidxe2x80x9d are employed interchangeably herein to refer to the acid form, i.e., 2-hydroxypropionic acid. The salt form of lactate is specifically referred to herein as a lactate salt, e.g., as either the sodium salt of lactic acid or sodium lactate.
The present invention also provides acid-tolerant homolactic bacteria. The acid-tolerant homolactic bacteria are generally capable of producing at least about 25 g/L free lactic acid at an incubation temperature of at least about 40xc2x0 C. Another embodiment of the present acid-tolerant bacteria is capable of producing at least about 50 g/L lactate at a temperature above about 40xc2x0 C. and an average incubation pH of no more than about 4.2. Typically, the acid-tolerant bacteria is capable satisfying both of these measures of lactate productivity.